1. Field of the Invention
The present invention is related to a substrate onto which bioactive substances can be favorably immobilized. The present invention is also related to a method for producing the substrate. Further, the present invention is related to a biosensor chip and a bioreactor chip equipped with the substrate.
2. Description of the Related Art
Presently, measurements that utilize intermolecular interactions, such as immune reactions in clinical tests, are being performed. Several techniques that do not require complex operations or labeling substances, and are capable of detecting variations in the bonding amounts of measurement target substances at high sensitivity, are being utilized. Examples of these techniques include: the Surface Plasmon Resonance (SPR) measurement technique, the quartz crystal microbalance (QCM) measurement technique, and a technique that utilizes the functional surfaces of gold colloid particles to superfine particles. In all of these techniques, surfaces on which bioactive substances are immobilized are necessary. This will be explained, using Surface Plasmon Resonance (SPR) as an example.
Commonly, measurement chips which are utilized to measure bioactive substances comprise: a transparent substrate (a glass plate, for example); a metal film formed by vapor deposition on the transparent substrate; and a thin film having functional groups onto which bioactive substances such as proteins can be immobilized; stacked in this order. Bioactive substances are immobilized onto the surface of the metal film via the functional groups. Specific bonding reactions between the bioactive substances and targets of measurement are measured, to analyze the interaction among biomolecules. Accordingly, improvements in bonding between the bioactive substances and the target measurement substances are desired in measurement chips.
SAM's (Self Assembling Monolayers), which have a constant regularity formed by mechanisms of the film material itself, without detailed control being exerted from the exterior, are examples of a thin film of a measurement chip that has functional groups capable of immobilizing bioactive substances. For example, “High-Affinity Chelator Thiols for Switchable and Oriented Immobilization of Histidine-Tagged Proteins: A Generic Platform for Protein Chip Technologies”, A. Tinazli et al., Chem. Eur. J., Vol. 11, pp. 5249-5259, 2005 discloses a technique by which SAM's are formed by a reagent having multivalent chelate thiol residues. In addition, “Production of Histidine Tagged Protein Arrays and Label Free Interaction Observations”, M. Kyo and T. Natsume, Toyobo Life Science Magazine, Vol. 77, pp. 15-16, 2004, discloses a technique for producing a two dimensional NTA film, by producing a SAM having carbonic acid at the ends thereof, then modifying the SAM with NTA in water.
However, the SAM disclosed in Chem. Eur. J. is a single component SAM formed by bis-NTA (bis-Nitrilotriacetic acid) or a mixed SAM formed by bis-NTA and a reagent having an OH group at the ends thereof. Therefore, the ends of the SAM become bulky, the SAM cannot be packed onto a metal film in an organized state, and gaps and defects in the SAM are likely to occur on the metal film. Therefore, there is a problem that non specific adsorption onto the metal film cannot be suppressed. In order to improve specific bonding between bioactive substances and detection target substances, it is effective to hold the bioactive substances at many points. However, because the NTA disclosed in Chem. Eur. J. is a bulky functional group, the SAM becomes rigid and not capable of moving flexibly. Accordingly, it is difficult for metal to coordinately bond with the bioactive substances at many points, and there is a problem that the bioactive substances cannot be stably immobilized at many points. Meanwhile, the modification method disclosed by M. Kyo and T. Natsume is not capable of binding NTA at a density sufficient to induce bonding at multiple points.